microRNAs (miRs) are small non-coding RNAs that regulate protein expression by destabilization and/or translational inhibition of target messenger RNAs (mRNAs). Like mRNAs, expression of miRs is regulated in cardiac hypertrophy and heart failure, fine-tuning cardiomyocyte function in response to immediate physiological demands. Single miRs tend to regulate numerous effectors within the same functional pathway, producing a coherent physiological response via multiple parallel perturbations, and are therefore attractive therapeutic targets. However, targeting of dozens or hundreds of different mRNAs by one miR, together with regulated expression of miRs and mRNAs in cardiac disease, complicates delineation of specific miR functions relevant to heart disease. We have developed new techniques combining RNA sequencing on massively parallel next generation platforms with in vivo miR programming of cardiac RNA-induced signaling complexes to overcome this problem. Additionally, we have identified common sequence variants and rare mutations within the mature miR sequence of 30 human miRs and demonstrated that both seed sequence and non-seed sequence miR variants can alter mRNA targeting. Accordingly, we hypothesize that miR effects on the normal and diseased heart are determined by the levels of expressed miRs, the levels of expressed mRNAs, and the efficiency of miR-mRNA pairing as determined by sequence complementarity, and that miR sequence variations further alter miR effects, independent of miR and mRNA expression levels. To examine these hypotheses we propose the first studies to systematically and comprehensively define important miR-mRNA pairing events and determine their functional consequences in genetically programmed mouse hearts under different pathophysiological conditions. We will follow with studies to determining the consequences of human miR sequence variants and mutations on cardiac miR-mRNA targeting, target protein expression, and cardiac structure and function. We are uniquely positioned to achieve these goals by combining our expertise in human genomics, RNA sequencing, and generation and analysis of murine genetic models. It is our long-term goal to integrate studies of human gene variation and cardiac disease with in vitro cell-based and in vivo murine experimental systems to fully understand the impact of miRs on the heart.